JP7287634B2 - Method for producing purified platelets - Google Patents

Description

TECHNICAL FIELD The present invention relates to a method for producing purified platelets from a culture of megakaryocyte cells, and the like.

Platelet preparations are administered to patients who exhibit massive bleeding during surgery or injury, or bleeding tendencies associated with decreased platelets after treatment with anticancer agents, for the purpose of treatment and prevention of such symptoms. Currently, production of platelets relies on blood donations by healthy volunteers. However, in Japan, the number of blood donors is declining due to the demographic structure, and it is estimated that there will be a shortage of about 1 million blood donations by 2027. Therefore, a stable supply of platelets is an important issue in the art.

In addition, conventional platelets are at high risk of bacterial contamination, which can lead to serious infections after platelet transplantation. Therefore, safer platelet preparations are always required in clinical practice. In order to meet the needs, today, a method for producing platelets from in vitro cultured megakaryocyte cells is being developed.

In rare cases, transfusion side effects (urticaria, anaphylactic reaction, etc.) may occur when platelet products are transfused. One of the reasons for this is considered to be plasma contained in platelet preparations. Therefore, in order to prevent transfusion side effects, a method has been developed in which the plasma in the platelet preparation is replaced with an artificially prepared liquid (washing and preserving liquid). For example, there is a method of washing platelets using a centrifugal separator equipped with a separation bowl in order to wash platelets in a platelet concentrate. The platelet concentrate referred to here is obtained by removing most of the leukocytes from blood components and suspending the collected platelets in plasma.

On the other hand, when megakaryocyte cells are cultured in vitro to produce platelets, it is necessary to separate and concentrate platelets from the megakaryocyte cells. Since the two have the same surface marker, they cannot be separated by a cell sorter. A method by centrifugation using However, in the method using a filter or a hollow fiber membrane, platelet bioactivity is lost due to surface protein damage, etc., and the recovery rate is as low as about 10%. In addition, in the method using centrifugation, since a low centrifugation speed is adopted so as not to deteriorate the function, the removal rate of megakaryocyte cells is low, the platelet recovery rate is only about 10%, and the amount that can be purified at one time is not centrifuged. The problem is that the volume of the tube is limited.

An object of the present invention is to provide a method for producing high-quality purified platelets by separating and purifying a large amount of platelets at once from a culture of megakaryocyte cells at a high recovery rate.

The present inventors, as a result of repeated studies to solve the above problems, after centrifuging the culture of megakaryocyte cells at a predetermined centrifugal force, the liquid component obtained by the centrifugal treatment higher centrifugal force By centrifuging once again, it was found that high-quality platelets can be purified with a high recovery rate from the culture of megakaryocyte cells.

That is, the present invention
[1] A method for producing purified platelets from a culture of megakaryocyte cells,
a first centrifugation step of centrifuging the culture at a centrifugal force of 150×g to 550×g;
a second centrifugation step of centrifuging the liquid component recovered in the first centrifugation step with a centrifugal force of 600 x g to 4000 x g;
[2] The centrifugation step is
A rotatable separation bowl having an inner wall to which a substance having a large specific gravity adheres according to centrifugal force, an outlet for discharging the liquid component after separation, and a recovery means for recovering the liquid component that has flowed out from the outlet. and the method according to [1], which is carried out by centrifugation.
[3] After the second centrifugal separation step, a washing step of adding a washing liquid to the separation bowl and rotating it;
After the washing step, a platelet recovery step of adding a recovery liquid and rotating;
The method according to [2], comprising;
[4] The method according to [2] or [3], wherein the first centrifugation step is to inject the culture into the separation bowl by gravity;
[5] The megakaryocyte culture is
A step of forcibly expressing an oncogene and a polycomb gene in cells that are undifferentiated from megakaryocyte cells;
a step of forcibly expressing the Bcl-xL gene in the cell;
a step of canceling all the forced expression;
The method according to any one of [1] to [4];
[6] A method for producing a blood product, comprising the step of mixing platelets purified by the method according to any one of [1] to [3] with other components;
Regarding.

According to the method of the present invention, high-quality platelets can be purified from megakaryocyte cell cultures at a high recovery rate and in large quantities at once, so a large amount of safe platelet preparations with low risk of bacterial contamination can produce and supply.

FIG. 1 shows the results of measuring platelet counts before and after purification by the purification method according to the present invention. FIG. 2 shows the results of measuring the physiological activity of platelets purified by the purification method according to the present invention. FIG. 3 shows the results of measuring the ratio of abnormal platelets after purification by the purification method according to the present invention. FIG. 4 shows the results of measuring the physiological activity of platelets 6 days after purification by the purification method according to the present invention. FIG. 5 shows the results of measuring the ratio of abnormal platelets 6 days after purification by the purification method according to the present invention.

The method for producing platelets according to the present invention includes a step of centrifuging a sample containing megakaryocyte cells two or more times with different centrifugal forces. For example, the first centrifugation step can be performed at a centrifugal force of about 150×g to about 550×g and the second centrifugation step can be performed at a centrifugal force of about 600×g to about 4000×g. In the centrifugal separation step, a rotatable separation bowl having an inner wall to which a substance having a large specific gravity adheres according to centrifugal force and an outlet for discharging the liquid component after separation,
Centrifugal separation provided with recovery means for recovering the liquid component that has flowed out from the outlet may also be used.

In the centrifugal separator, when the liquid mixture is injected while the separation bowl is rotated about an axis that passes through the center of the bottom surface and is perpendicular to the bottom surface, the component with a large specific gravity adheres to the inner wall of the separation bowl according to the centrifugal force.・The component with low specific gravity remains in the liquid.

A liquid containing a component with a low specific gravity is recovered by the recovery means. The collection means is composed of, for example, a tube connected to the outlet of the separation bowl and a collection bag exchangeably connected to the tube. The collection bag is not particularly limited as long as it does not affect the quality of platelets, but a commercially available blood or blood component storage bag may be used.

The centrifugal separation device performs centrifugal separation while injecting a liquid mixture into the separation bowl at a predetermined speed, and at the same time, the separated liquid can be recovered by the recovery means. The mixture can be separated continuously.

As the centrifugation device used in the method for producing purified platelets according to the present invention, for example, the device disclosed in JP-A-2005-296675 and the device disclosed in JP-A-7-284529 can be used. can. Moreover, commercially available centrifugal separators used for separating blood components and commercially available apparatuses conventionally used for washing platelets in concentrated platelet preparations can also be used. For example, ACP215 from HAEMONETICS and COBE2991 from Terumo may be used.

In certain embodiments, the first centrifugation step may be performed by spinning the separation bowl at a centrifugal force of about 150 xg to about 550 xg. The centrifugal force is preferably about 160×g to about 500×g, more preferably about 170×g to about 400×g, even more preferably about 180×g to about 300×g. By performing centrifugation with the centrifugal force in this range, the megakaryocyte cells in the culture adhere and accumulate inside the separation bowl. If the centrifugal force is increased beyond this point, shear stress is applied to the platelets, causing physiological activity of the platelets to cause aggregation.
Since the following relationship holds between the centrifugal force (g), rotation speed (rpm), and radius of rotation (cm), the rotation speed can be determined according to the size of the rotating bowl to be used.
Centrifugal force (g) = 1119 x Radius of rotation (cm) x (Number of revolutions (rpm)) ² x 10 ^-8

In this step, a platelet stock solution may be added to the culture of megakaryocyte cells. Examples thereof include biological product standard blood preservation solution A (Acid-Citrate-Dextrose; ACD-A). ACD-A has a blood/platelet anticoagulant action and also functions as a glucose supply source, which is an energy source for platelets.

During the first centrifugation step, the method of injecting the culture of megakaryocyte cells into the separation bowl is not particularly limited, and a pump provided in the device may be used, and the container containing the culture and the separation bowl may be connected by a tube, the container may be hung high, and the culture may be allowed to fall naturally into the bowl through the tube. Infusion rates can be, for example, from about 50 ml/min to about 150 ml/min, from about 80 ml/min to about 130 ml/min, or about 100 ml/min.

The first centrifugation step can be performed at room temperature. The duration of the first centrifugation step can be the volume of the culture divided by the injection rate of the culture, or longer.
During the first centrifugation step, the platelets remain in solution and are collected by the collection means.

The second centrifugation step of the method for purifying platelets according to the present invention is a step of separating platelets from the liquid component collected in the first centrifugation step.
After the first centrifugation step, the separation bowl is replaced or washed, and then the liquid component recovered in the first centrifugation step is injected while rotating the separation bowl. Injection can be performed similarly to the first centrifugation step. The collection bag used in the first centrifugation step may be suspended in a high position and injected into the separation bowl by gravity through the tube.

In certain embodiments, the second centrifugation step may be performed by spinning the separation bowl at a centrifugal force of about 600 xg to about 4000 xg. Centrifugal force may be preferably about 800×g to about 3000×g, more preferably about 1000×g to about 2000×g. By performing centrifugal separation with the centrifugal force within this range, platelets can adhere and accumulate on the inner wall of the separation bowl without losing their physiological activity.
A second centrifugation step can be performed at room temperature. The time of the first centrifugation step can be equal to or longer than the volume of the liquid component collected in the first centrifugation step divided by the injection rate of the liquid.
The liquid component separated in the second centrifugal separation step is recovered by the recovery means and discarded.

In another embodiment, a washing step may be performed after the second centrifugation step. In the second centrifugation step, the medium and additives as well as the platelets adhere and accumulate on the inner wall of the separation bowl. It is a washing step for removing this medium.
In the washing step, a wash solution is added into the separation bowl and spun with a centrifugal force of about 600×g to about 3600×g. A bicarbonate Ringer's solution, such as Bicarbon, may be used as the cleaning liquid. Also, a platelet preservation solution may be added to Bicarbon. The platelet preservation solution may be added with ACD solution, for example. When adding the cleaning liquid to the separation bowl, the cleaning liquid is injected at a constant speed. At this time, the platelets are kept attached to the inner wall of the separation bowl, and the washing liquid containing the culture medium and additives is collected by the collecting means.

In another embodiment, the washing step may be followed by a platelet recovery step. In the recovery step, a recovery liquid is added to recover platelets adhering to the inner wall of the separation bowl, and the bowl is spun with a centrifugal force of about 600×g to about 3600×g. This causes the platelets to be shaken off the separation bowl and float in the collected liquid. Bicarbon, for example, can be used as the recovery liquid, and is injected into the separation bowl at a constant speed. 5% ACD may be added to bicarbon. A recovery liquid containing platelets flows out from the outlet of the separation bowl and is recovered in a recovery means such as a recovery bag. This can also be used as the final product.

As used herein, the term “megakaryocyte” refers to the largest cell existing in the bone marrow in vivo, and is characterized by releasing platelets. The megakaryocyte is characterized by positive cell surface markers CD41a, CD42a, and CD42b, and is selected from the group consisting of CD9, CD61, CD62p, CD42c, CD42d, CD49f, CD51, CD110, CD123, CD131, and CD203c. It may also express a marker that is Megakaryocyte cells, when multinucleated (polyploidized), have 16 to 32 times the genome of normal cells. As used herein, the term "megakaryocyte" simply includes both multinucleated megakaryocyte and pre-multinucleated megakaryocyte as long as it has the above characteristics. "Megakaryocyte cells before multinucleation" is also synonymous with "immature megakaryocyte cells" or "proliferative megakaryocyte cells".

Megakaryocyte cells can be obtained by various known methods. Non-limiting examples of methods for producing megakaryocyte cells include the method described in International Publication No. 2011/034073. In this method, an immortalized megakaryocyte cell line that proliferates indefinitely can be obtained by forcibly expressing the oncogene and the polycomb gene in "cells that are undifferentiated from megakaryocyte cells". In addition, according to the method described in International Publication No. 2012/157586, in "cells undifferentiated from megakaryocyte cells", immortalized megakaryocyte cell lines can also be obtained by forced expression of apoptosis suppressor genes. . These immortalized megakaryocyte cell lines undergo multinucleation and release platelets by canceling forced gene expression.

In order to obtain megakaryocyte cells, the methods described in the above documents may be combined. In that case, forced expression of the oncogene, polycomb gene and apoptosis suppressor gene may be performed simultaneously or sequentially. For example, an oncogene and a polycomb gene may be forcibly expressed, the forced expression may be suppressed, then an apoptosis suppressor gene may be forcedly expressed, the forced expression may be suppressed, and multinucleated megakaryocyte cells may be obtained. Alternatively, the oncogene, the polycomb gene and the apoptosis suppressor gene can be forced to express simultaneously, and the forced expression can be suppressed simultaneously to obtain multinucleated megakaryocyte cells. First, the oncogene and Polycomb gene are forcibly expressed, then the apoptosis suppressor gene is forcibly expressed, and the forced expression is simultaneously suppressed to obtain multinucleated megakaryocyte cells.

As used herein, the term “cells undifferentiated from megakaryocyte cells” refers to cells that have the ability to differentiate into megakaryocytes, and that are cells in various stages of differentiation from hematopoietic stem cell lineage to megakaryocyte cells. Non-limiting examples of cells less differentiated than megakaryocytes include hematopoietic stem cells, hematopoietic progenitor cells, CD34-positive cells, and megakaryocyte/erythroid progenitor cells (MEP). These cells can be obtained by, for example, isolating from bone marrow, umbilical cord blood, or peripheral blood, or can be obtained by inducing differentiation from pluripotent stem cells such as ES cells and iPS cells, which are more undifferentiated cells. can.

As used herein, the term "oncogene" refers to a gene that induces canceration of cells in vivo. genes, RAS family genes, RAF family genes, protein kinase family genes such as c-Kit, PDGFR, and Abl.

As used herein, "polycomb gene" is known as a gene that negatively regulates the CDKN2a (INK4a/ARF) gene and functions to avoid cell senescence (Ogura et al., Regenerative Medicine vol.6, No. 4, pp26-32; Jseus et al., Jseus et al., Nature Reviews Molecular Cell Biology vol.7, pp667-677, 2006; Proc. Natl. Acad. Sci. ). Non-limiting examples of Polycomb genes include BMI1, Mel18, Ring1a/b, Phc1/2/3, Cbx2/4/6/7/8, Ezh2, Eed, Suz12, HADC, Dnmt1/3a/3b .

As used herein, the term "apoptosis suppressor gene" refers to a gene that has the function of suppressing cell apoptosis, and includes, for example, BCL2 gene, BCL-xL gene, Survivin gene, and MCL1 gene.

Forced expression of genes and release of forced expression are described in WO 2011/034073, WO 2012/157586, WO 2014/123242 or Nakamura S et al, Cell Stem Cell. 14, 535-548, 2014 can be carried out by the method described in , other known methods, or methods based thereon.

As used herein, "platelets" are one of the cellular components in blood and are characterized by being CD41a-positive and CD42b-positive. Platelets play an important role in thrombus formation and hemostasis, and are also involved in post-injury tissue regeneration and the pathophysiology of inflammation. When platelets are activated by bleeding or the like, receptors for cell adhesion factors such as integrin αIIBβ3 (glycoprotein IIb/IIIa; complex of CD41a and CD61) are expressed on the membrane. As a result, platelets aggregate with each other and fibrin is coagulated by various blood coagulation factors released from the platelets to form a thrombus and promote hemostasis.

Platelets purified by the method of the present invention are of high quality. As used herein, the term “high-quality purified platelets” refers to platelets from which megakaryocyte cells have been substantially removed so that physiological activity per fraction is maintained at a high level and abnormal platelets are sufficiently low.

Physiological activities of platelets can be measured and evaluated by known methods. For example, an antibody to PAC-1 that specifically binds to integrin αIIBβ3 on activated platelet membranes can be used to measure the amount of activated platelets. Similarly, CD62p (P-selectin), which is a platelet activation marker, may be detected with an antibody to measure the amount of activated platelets. For example, it can be performed by using flow cytometry, performing gating with an antibody against the activation-independent platelet marker CD61 or CD41, and then detecting binding of an anti-PAC-1 antibody or an anti-CD62p antibody. These steps may be performed in the presence of adenosine diphosphate (ADP).

In addition, evaluation of platelet function can also be performed by observing whether or not platelets bind to fibrinogen in the presence of ADP. Binding of platelets to fibrinogen results in the activation of integrins that are required early in thrombus formation.
Furthermore, evaluation of platelet function can also be performed by a method of visualizing and observing in vivo thrombus forming ability, as shown in FIG. 6 of WO 2011/034073.

On the other hand, when the platelet CD42b expression rate is low or when the annexin V positive rate is low, the platelets are evaluated as degraded or abnormal. These platelets do not have sufficient thrombogenic or hemostatic function and are not clinically useful.

As used herein, “platelet deterioration” refers to a decrease in CD42b (GPIbα) on the platelet surface. Degraded platelets therefore include platelets with decreased CD42b expression and platelets in which the extracellular region of CD42b has been cleaved by the shedding reaction. When CD42b on the platelet surface is lost, it becomes impossible to associate with von Willebrand factor (VWF), and as a result, the blood coagulation function of platelets is lost. Degradation of platelets can be evaluated using the CD42b negative rate (or CD42b negative particle count) relative to the CD42b positive rate (or CD42b positive particle count) in the platelet fraction as an index. The higher the CD42b-negative rate relative to the CD42b-positive rate, or the greater the CD42b-negative particle count relative to the CD42b-positive particle count, the worse the platelets are. The CD42b positive rate means the percentage of platelets that can bind to anti-CD42b antibody among the platelets contained in the platelet fraction, and the CD42b negative rate means that among the platelets contained in the platelet fraction, anti-CD42b antibody binds. means the percentage of platelets that do not.

As used herein, the term "abnormal platelets" refers to platelets in which the negatively charged phospholipid, phosphatidylserine, is exposed from the inside to the outside of the lipid bilayer. It is known that in vivo, phosphatidylserine is exposed on the surface of platelets upon platelet activation, and many blood coagulation factors bind to the surface, thereby amplifying the blood coagulation cascade reaction. On the other hand, in abnormal platelets, a large amount of phosphatidylserine is always exposed on the surface, and when such platelets are administered to patients, they cause excessive blood coagulation reactions, leading to serious pathological conditions such as disseminated intravascular coagulation. may lead to Since annexin V binds to phosphatidylserine, phosphatidylserine on the platelet surface can be detected using a flow cytometer using the amount of fluorescence-labeled annexin V bound as an indicator. Therefore, the amount of abnormal platelets can be assessed by the annexin V positive rate in the platelet fraction, ie the percentage or number of platelets to which annexin binds. The higher the annexin V positive rate or the higher the annexin V particle count, the more abnormal platelets.

Culture conditions for megakaryocyte cells in the present invention can be normal conditions. For example, the temperature can be from about 35°C to about 42°C, from about 36°C to about 40°C, or from about 37°C to about 39°C, and can be 5-15% _CO2 and/or 20% _O2 . .

The medium for culturing megakaryocyte cells is not particularly limited, and a known medium suitable for producing platelets from megakaryocyte cells or a medium based thereon can be used as appropriate. For example, a medium used for culturing animal cells can be prepared as a basal medium. Examples of basal media include IMDM medium, Medium 199 medium, Eagle's Minimum Essential Medium (EMEM) medium, αMEM medium, Dulbecco's modified Eagle's Medium (DMEM) medium, Ham's F12 medium, RPMI 1640 medium, Fischer's medium, and Neurobasal Medium (Life Technologies). ) and mixed media thereof.

The medium may contain serum or plasma, or may be serum-free. Optionally, the medium contains e.g. albumin, insulin, transferrin, selenium, fatty acids, trace elements, 2-mercaptoethanol, thiolglycerol, monothioglycerol (MTG), lipids, amino acids (e.g. L-glutamine), ascorbic acid , heparin, non-essential amino acids, vitamins, growth factors, small compounds, antibiotics, antioxidants, pyruvate, buffers, inorganic salts, cytokines, and the like. Cytokines are proteins that promote blood cell lineage differentiation. ) supplements, ADAM inhibitors, and the like. A preferred medium in the present invention is IMDM medium containing serum, insulin, transferrin, serine, thiolglycerol, ascorbic acid and TPO. Furthermore, it may contain SCF, and may further contain heparin. Each concentration is also not particularly limited, but for example, TPO can be about 10 ng/mL to about 200 ng/mL, or about 50 ng/mL to about 100 ng/mL, and SCF can be about 10 ng/mL to about 200 ng. /mL, or about 50 ng/mL, and heparin can be about 10 U/mL to about 100 U/mL, or about 25 U/mL. Phorbol esters (eg phorbol-12-myristate-13-acetate; PMA) may be added.

When serum is used, human serum is preferred. Also, instead of serum, human plasma or the like may be used. According to the method of the present invention, platelets equivalent to those obtained using serum can be obtained using these components.

A drug-responsive gene expression induction system such as the Tet-on (registered trademark) or Tet-off (registered trademark) system may be used for forced gene expression and release thereof. In that case, in the forced expression step, the forced expression can be suppressed by adding a corresponding drug such as tetracycline or doxycycline to the medium and removing them from the medium.

Since the culture step of megakaryocyte cells in the present invention is performed by suspension culture, it can be performed without feeder cells.

The "megakaryocyte cell culture" according to the present invention is a culture obtained in the above-described culture step, and includes megakaryocyte cells and a culture solution containing various additives.

The present invention also includes platelets purified by the method of the present invention.

The method for producing a blood product according to the present invention includes the steps of producing a platelet product by the method according to the present invention and mixing the platelet product with other components. Other components include, for example, red blood cells.
The invention also includes blood products purified by this method.
In addition, other ingredients that contribute to cell stabilization may be added to platelet preparations and blood preparations.

The disclosures of all patent and non-patent literature cited herein are hereby incorporated by reference in their entirety.

EXAMPLES The present invention will be specifically described below based on examples, but the present invention is not limited to these. Persons skilled in the art can modify the present invention in various ways without departing from the meaning of the invention, and such modifications are also included in the scope of the present invention.

1. Generation of immortalized megakaryocyte cells
1-1. Preparation of hematopoietic progenitor cells from iPS cells From human iPS cells (TKDN SeV2: iPS cells derived from human fetal skin fibroblasts established using Sendai virus), Takayama N., et al. J Exp Med. (2010), differentiation culture into blood cells was performed. That is, human ES/iPS cell colonies were co-cultured with C3H10T1/2 feeder cells for 14 days in the presence of 20 ng/mL VEGF (R&D SYSTEMS) to prepare hematopoietic progenitor cells (HPC). The culture conditions were 20% O ₂ and 5% CO ₂ (same conditions below unless otherwise specified).

1-2. Gene transfer system A lentiviral vector system was used as the gene transfer system. The lentiviral vector is a Tetracycline-regulated Tet-on® gene expression induction system vector. It was produced by recombining the mOKS cassette of LV-TRE-mOKS-Ubc-tTA-I2G (Kobayashi, T., et al. Cell 142, 787-799 (2010)) with c-MYC, BMI1, and BCL-xL. LV-TRE-c-Myc-Ubc-tTA-I2G, LV-TRE-BMI1-Ubc-tTA-I2G, and LV-TRE-BCL-xL-Ubc-tTA-I2G, respectively.
Virus particles were produced by transfecting 293T cells with the above lentiviral vector.
By infecting the target cells with such virus particles, the BMI1, MYC, and BCL-xL genes are introduced into the target cell's genomic sequence. These genes stably introduced into the genomic sequence can be forced to express by adding doxycycline (clontech #631311) to the medium.

1-3. Infection of hematopoietic progenitor cells with c-MYC and BMI1 viruses On a 6-well plate pre-seeded with C3H10T1/2 feeder cells, HPCs obtained by the above method were seeded at 5 x 10 ⁴ cells/well, and lentiviral method was used. Forced expression of c-MYC and BMI1. At this time, 6 wells were used for each type of cell line. That is, virus particles were added to the medium at an MOI of 20, respectively, and infected by spin infection (centrifugation at 32°C, 900 rpm for 60 minutes). This operation was performed twice every 12 hours.
The medium consists of basal medium (15% Fetal Bovine Serum (GIBCO), 1% Penicillin-Streptomycin-Glutamine (GIBCO), 1% Insulin, Transferrin, Selenium Solution (ITS-G) (GIBCO), 0.45mM 1-Thioglycerol (Sigma -Aldrich), 50ng/mL Human thrombopoietin (TPO) (R&D SYSTEMS), 50ng/mL Human A medium supplemented with Stem Cell Factor (SCF) (R&D SYSTEMS) and 2 µg/mL Doxycycline (Dox) (hereinafter referred to as differentiation medium) was further supplemented with Protamine at a final concentration of 10 µg/mL.

1-4. Preparation and maintenance culture of megakaryocyte self-propagating strain The day cMYC and BMI1 virus infection was performed by the above method as day 0 of infection, and as follows, by culturing cMYC and BMI1 transgenic megakaryocyte cells, megakaryocytes Each sphere autologous strain was generated. Forced expression of the BMI1 gene and c-MYC gene was performed by adding 1 μg/mL of doxycycline (clontech #631311) to the medium.

・From day 2 of infection to day 11 of infection Collect the virus-infected blood cells obtained by the above method by pipetting, centrifuge at 1200 rpm for 5 minutes to remove the supernatant, and add new differentiation medium. It was suspended and plated on fresh C3H10T1/2 feeder cells (6 well plate). Passaging was carried out by performing the same operation on day 9 of infection. After counting the number of cells, the cells were seeded on C3H10T1/2 feeder cells at 1×10 ⁵ cells/2 mL/well (6 well plate).

・Day 12 to day 13 of infection The same procedure as on day 2 of infection was performed. After counting the number of cells, the cells were seeded on C3H10T1/2 feeder cells at 3×10 ⁵ cells/10 mL/100 mm dish (100 mm dish).

・ On day 14 of infection, virus-infected blood cells were collected, and per 1.0 × 10 ⁵ cells, anti-human CD41a-APC antibody (BioLegend), anti-human CD42b-PE antibody (eBioscience), anti-human CD235ab-pacific blue (BioLegend) ) Antibody reaction was performed using 2 μL, 1 μL, and 1 μL of each antibody. After the reaction, it was analyzed using FACS Verse (BD). Cells with a CD41a positive rate of 50% or more on day 14 of infection were defined as megakaryocyte autologous strains.

1-4. BCL-xL Virus Infection into Self-Proliferating Megakaryocyte Strain BCL-xL was gene-introduced into the self-proliferating megakaryocyte strain on day 14 of infection by the lentiviral method. Virus particles were added to the medium at an MOI of 10, and infected by spin infection (centrifugation at 32°C, 900 rpm for 60 minutes). Forced expression of the BCL-xL gene was performed by adding 1 μg/mL of doxycycline (clontech #631311) to the medium.

1-5. Creation and maintenance culture of megakaryocyte immortalized strain/infection day 14-infection day 18 The megakaryocyte self-proliferating strain transfected with BCL-xL obtained by the above method is collected and centrifuged at 1200 rpm for 5 minutes. rice field. After centrifugation, the precipitated cells were suspended in a new differentiation medium and seeded onto new C3H10T1/2 feeder cells at 2×10 ⁵ cells/2 mL/well (6-well plate).

- Day 18 of infection: Passage After counting the number of cells, the cells were seeded at 3 x ¹⁰⁵ cells/10 mL/100 mm dish.

- Day 24 of infection: Passage After counting the number of cells, the cells were seeded at 1 x ¹⁰⁵ cells/10 mL/100 mm dish. Thereafter, subculture was performed every 4 to 7 days, and maintenance culture was performed.

On day 24 of infection, the BCL-xL gene-introduced megakaryocyte self-proliferation strain was collected, and anti-human CD41a-APC antibody (BioLegend), anti-human CD42b-PE antibody (eBioscience), anti-human CD42b-PE antibody (eBioscience) were applied per 1.0 × 10 ⁵ cells. After immunostaining with 2 μL, 1 μL, and 1 μL of human CD235ab-Pacific Blue (Anti-CD235ab-PB; BioLegend) antibody, respectively, analysis was performed using FACS Verse (BD). A strain with a positive rate of 50% or more was defined as an immortalized megakaryocyte cell strain. These cells, which were able to proliferate for 24 days or more after infection, were designated as the immortalized megakaryocyte cell line SeV2-MKCL.

The resulting SeV2-MKCL was statically cultured in a 10 cm dish (10 mL/dish). IMDM was used as a basal medium, and the following components were added (concentrations are final concentrations).
FBS (Sigma #172012 lot.12E261) 15%
L-Glutamin (Gibco #25030-081) 2mM
ITS (Gibco #41400-045) diluted 1:100
MTG (monothioglycerol, sigma #M6145-25ML) 450μM
Ascorbic acid (sigma #A4544) 50 μg/mL
Puromycin (sigma #P8833-100MG) 2 μg/mL
SCF (Wako Pure Chemical #193-15513) 50ng/mL
TPO-like agent 200ng/mL
The culture conditions were 37°C and 5% CO ₂ .

2. Platelet production Next, forced expression was released by culturing in a medium containing no doxycycline. Specifically, 1. The immortalized megakaryocyte cell line (SeV2-MKCL) obtained by the method of 1 was washed twice with PBS(-) and suspended in the following medium for platelet production. The cell seeding density was 1.0×10 ⁵ cells/mL.

Platelets were produced by culturing in platelet production medium for 6 days.

A medium for platelet production (platelet production medium) was prepared by adding the following components to IMDM as a basal medium (concentrations are final concentrations).
FBS 15%
L-Glutamin (Gibco #25030-081) 2mM
ITS (Gibco #41400-045) diluted 1:100
MTG (monothioglycerol, sigma #M6145-25ML) 450μM
Ascorbic acid (sigma #A4544) 50 μg/mL
SCF (Wako Pure Chemical #193-15513) 50ng/mL
TPO-like agent 200ng/mL
ADAM inhibitor 15 μM
SR1 750nM
ROCK inhibitor 10 μM

3. Purification of platelets 3-1. Megakaryocyte removal First, the waste bag of the ACP215 disposable set was replaced with a collection bag using a sterile conjugator. Hycalic IVH bag (Terumo HC-B3006A) was used as a collection bag.
Next, 2.4 L of culture medium containing megakaryocytes obtained in the platelet production process and produced platelets was prepared. 10% of ACD-A solution was added to 2.4 L of such culture solution. After that, the culture medium to which the ACD-A solution was added was injected into the cell bag. Hycalic IVH bag (Terumo HC-B3006A) was used as the cell bag.
The cell bag containing culture medium was then bonded to the ACP215 disposable set using a sterile mating device.
The ACP215 was started in service mode and the centrifugation speed was set at 2000 rpm (223.8 xg).
The ACP215 disposable set was set on the ACP215, and the cell bag containing the medium was placed on the stand.
ACP215 was started and the medium in the cell bag was pumped into the separation bowl at approximately 100 mL/min. The eluate eluted from the bowl was collected in a collection bag.
After adding the entire amount of medium in the cell bag to the separation bowl, an additional 500 mL of wash solution was added.
After injecting the wash solution into the separation bowl, the centrifugation was stopped and the circulation bag containing the recovery solution (including platelets) was separated using a tube sealer.

3-2. Concentration, Washing, Formulation (1) Concentration Process A collection bag containing a collection liquid (containing platelets) was joined to a new ACP215 disposable set using a sterile joining device.
Booted ACP215 in normal mode. WPC was selected as the program setting, and the ACP215 disposable set to which the collection bag described above was joined was set according to the instructions of the device. The recovery bag containing the recovery liquid was placed on a stand.
Next, the centrifugation speed of ACP215 was changed to 5000 rpm (1398.8×g) and centrifugation was started.
When the recovery fluid into the separation bowl began to pour, the auto-injection was changed to manual injection. Specifically, the collected liquid was injected into the separation bowl at about 100 mL/min. After adding the entire amount of the collected liquid to the separation bowl, 500 mL of washing liquid was added.
(2) Washing step Washing was performed with 2000 mL of washing solution according to the ACP215 program.
(3) Formulation
200 mL of washed platelets were collected into a platelet product bag according to the ACP215 program.

4. Measurement of megakaryocyte count, platelet count, platelet physiological activity, and abnormal platelets
4-1. Measurement method The megakaryocyte count, platelet count, platelet physiological activity, abnormal platelets, etc. in the purified platelet preparation were measured using a flow cytometer.
Megakaryocyte count, platelet count, platelet physiological activity measurement, 900 μL of diluent was added to a 1.5 mL microtube, 100 μL of megakaryocyte cell culture or platelet purification recovery was added and mixed. 200 µL of the resulting solution was dispensed into FACS tubes, and a labeled antibody was added for staining.
In the measurement of abnormal platelets, dispensed 100μL of megakaryocyte culture or collected after platelet purification into FACS tubes, stained by adding labeled antibody and protein, annexin V binding buffer just before flow cytometer analysis (BD) was diluted 5-fold and analyzed.
The following antibodies were used.
(1) Measurement of megakaryocyte count and platelet count
1.0 μL anti-CD41a antibody APC-labeled (Bio Legend 303710)
1.0 μL anti-CD42a antibody PB-labeled (eBioscience 48-0428-42)
1.0 μL anti-CD42b antibody PE-labeled (Bio Legend 303906)
(2) Measurement of physiological activity of platelets
0.5 μL anti-CD42a antibody PB-labeled (eBioscience 48-0428-42)
0.5 μL anti-CD42b antibody PE-labeled (Bio Legend 303906)
0.5 μL anti-CD62p antibody APC-labeled (Bio Legend 304910)
10 μL anti-PAC-1 antibody FITC-labeled (BD 303704)
(3) Measurement of abnormal platelet count
1.0 μL anti-CD41a antibody APC-labeled (Bio Legend 303710)
1.0 μL anti-CD42b antibody PE-labeled (Bio Legend 303906)
5 μL Annexin V FITC-labeled (BD 556419)

精製前
（2600mL）
精製後
（200mL）
収率（%）

血小板
2.40×10¹⁰
7.41×10⁹
30.8

巨核球数
6.91×10⁸
1.60×10⁷
3.3

4-2. Measurement results of megakaryocyte count and platelet count
The number of CD41a-positive and CD42b-positive particles was defined as the platelet count, and the number of positive particles was defined as the megakaryocyte cell count. The number of platelets and the number of megakaryocytes contained in the culture before purification (culture cultured for 6 days using Versus) and the recovered product after purification were measured.
The results are shown in the table below and in FIG.

Before purification (2600mL)
After purification (200mL)
yield(%)

platelet
2.40×10 ¹⁰
7.41× ¹⁰⁹
30.8

Megakaryocyte count
6.91× ¹⁰⁸
1.60× ¹⁰⁷
3.3

From Table 1, the platelet recovery rate was 30.8%. In addition, the number of megakaryocytes decreased to 2.3% before purification (97.7% megakaryocyte removal rate). Compared to the conventional method (method using a filter or centrifugation method using a centrifugal tube), the platelet recovery rate increased threefold.

4-3. Measurement results of platelet physiological activity Stimulation of platelets was performed with PMA 0.2 μM (Phorbol 12-myristate 13-acetate, sigma #P1585-1MG) or ADP 100 μM (sigma #A2754) and Thrombin 0.5 U/mL (sigma). It was done at room temperature. Thirty minutes after the start of stimulation, measurement was performed using BD's FACSverse.
PAC-1 positive rate and CD62p (p-selectin) before and after stimulation in the CD42a positive platelet fraction
The positive rate was measured, and physiological activity was comparatively evaluated.
The results are shown in FIG. PAC-1-positive and CD62p (p-selectin)-positive cells increased after stimulation, confirming that the purified platelets maintained high physiological activity.

4-4. Measurement result of abnormal platelets The number of annexin V-positive particles was defined as the number of abnormal platelets. The results are shown in FIG.
The annexin V positive rate was as low as 14.5%, and platelet abnormalities were sufficiently suppressed.

4-5. Measurement results of physiological activity and abnormalities of platelets 6 days after purification 3-2. Physiological activities and abnormalities of platelets 6 days after purification were measured in the same manner as in 3-3.
The results are shown in FIGS. 4 and 5. FIG.
Even after 6 days of purification, the physiological activity of platelets was maintained at a high level, and the number of abnormal platelets was sufficiently low.

Claims (6)

A method for producing purified platelets from a culture of megakaryocyte cells,
In a rotatable separation bowl, the megakaryocyte cells are removed from the culture by centrifuging the culture at a centrifugal force of 150 × g to 550 × g, and the liquid component containing platelets separated from the megakaryocytes is a step of recovering;
and purifying the platelets in the liquid component by centrifuging the liquid component collected in the step with a centrifugal force of 600×g to 3000×g in a rotatable separation bowl. The centrifugation is
a rotatable separation bowl having an inner wall for adhering a substance having a large specific gravity according to centrifugal force, and an outlet for discharging the separated liquid component;
2. The method according to claim 1, wherein the method is carried out in a centrifugal separator comprising a recovery means for recovering the liquid component discharged from said outlet. The step of purifying platelets includes, after a centrifugal separation step with a centrifugal force of 600 x g to 3000 x g , a washing step of adding a washing solution to the separation bowl and rotating it;
After the washing step, a platelet recovery step of adding a recovery liquid and rotating;
3. The method of claim 2, comprising: The method according to claim 2 or 3, wherein the centrifugation step at a centrifugal force of 150xg to 550xg pours the culture into the separation bowl by gravity. The megakaryocyte culture is
A step of forcibly expressing an oncogene and a polycomb gene in cells that are undifferentiated from megakaryocyte cells;
a step of forcibly expressing the Bcl-xL gene in the cell;
a step of canceling all the forced expression;
The method according to any one of claims 1 to 4, which is obtained from A method for producing a blood product, comprising the step of mixing purified platelets produced by the method according to any one of claims 1 to 5 and a platelet storage solution.

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